The proposed research builds on recent extensions to our hardware and software that allows, in a generalized resolution overlapping fashion, 3- dimensional reconstructions of subcellular structures at both the light and electron microscopic levels. Recent progress makes possible collection of in vivo 3-dimensional time dependent multiple wavelength image information, with subsequent quantitative analysis, to provide insights into chromosome structure. We apply this methodology to the analysis of higher-order chromosome structure and function using the unique interphase polytene chromosomes of Drosophila melanogaster. We focus on cytologically identified polytene chromosome bands, interbands and puffs (transcriptionally active, morphologically distinctive chromosome sites). We utilize Drosophila metamorphosis, triggered by the insect hormone ecdysone, as a way to target a characteristic transcriptional cascade. The specific aims of this proposal are to: A) Continue the determination of the 3-dimensional structure of specific polytene chromosome bands, interbands and puffs using electron microscope tomography. B) Study the in vivo structural dynamics (quantitative motion analysis) of pulse labeled DNA replication sites within polytene chromosomes. C) Study the in vivo structural dynamics of ecdysone induced puff formation and regression. D) Study the structural dynamics, on polytene chromosome puffs, of proteins (ecdysone receptor and early puff encoded proteins) associated with the ecdysone response. E) Using imaging resonance energy transfer methods, determine the dynamics of specific protein-protein associations within puff structures.